A subpopulation of neuronal M4 mAChRs plays a critical role in modulating dopamine-dependent behaviors Initial studies with whole body M4 mAChR KO mice suggested that M4 mAChRs are involved in modulating the activity of the central dopaminergic system. The specific neuronal subpopulations involved in the interactions of the M4 mAChR with the central dopaminergic system have not been identified. Previous studies have shown that the M4 mAChR is widely distributed throughout the forebrain. Interestingly, M4 receptors are coexpressed with D1 dopamine receptors in a specific subset of striatal medium spiny neurons which contain GABA as the major neurotransmitter and give rise to the so-called striato-nigral pathway. To investigate the physiological relevance of this subpopulation of neuronal M4 mAChRs, we used Cre/loxP technology to generate mutant mice that lack M4 mAChRs only in D1 dopamine receptor-expressing cells. We then analyzed these newly generated mutant mice in a series of biochemical and behavioral studies, with a particular focus on behavioral responses that involve the dopaminergic system. Interestingly, the newly generated M4 mAChR mutant mice displayed several striking phenotypes, including enhanced hyperlocomotor activity and increased behavioral sensitization following treatment with psychostimulants. These behavioral changes were accompanied by specific biochemical and neurochemical changes. Moreover, the newly generated mutant mice displayed reduced cataleptic responses to first or second generation antipsychotic drugs. These novel observations indicate that M4 mAChRs expressed by D1 dopamine receptor-expressing neurons play a key role in modulating several key functions of the central dopaminergic system. Since an increase in dopaminergic neurotransmission has been implicated in the pathogenesis of schizophrenia, drug addiction, and several other CNS disorders, these findings should be of considerable therapeutic interest. Publication: Jeon J, Dencker D, Wrtwein G, Woldbye DP, Cui Y, Davis AA, Levey AI, Schtz G, Sager TN, Mrk A, Li C, Deng CX, Fink-Jensen A, Wess J. A subpopulation of neuronal M4 muscarinic acetylcholine receptors plays a critical role in modulating dopamine-dependent behaviors. J. Neurosci. 30, 2396-405, 2010. The following results were obtained in collaborative studies: 1. Using M1 and M4 mAChR KO mice as tools, we found that M1 and M4 receptor-dependent functions are involved in regulating sensorimotor gating mechanisms (prepulse inhibition). This finding suggests that muscarinic drugs targeting central M1 and/or M4 receptors may prove beneficial for the treatment of schizophrenia. Thomsen M, et al. Psychopharmacology (Berlin) 208, 401-416, 2010. 2. Behavioral studies with M1 and M4 mAChR KO mice demonstrated that central M1 receptors exert an inhibitory effect on the abuse-related effects of cocaine. This observation raises the possibility that M1 receptor agonists may prove beneficial for the treatment of cocaine addiction. Thomsen M, et al. J. Pharmacol. Exp. Ther. 332, 959-969, 2010. 3. We demonstrated that amphetamine-induced hyperactivity and dopamine release as well as amphetamine sensitization were enhanced in M5 mAChR KO mice. This finding further supports the concept that M5 receptor activity plays an important role in modulating the effects for drugs of abuse. Schmidt LS, et al. Psychopharmacology (Berlin) 207, 547-558, 2010. 4. Using tissues from mAChR KO mice, we demonstrated that striatal mAChRs mediate variable control of dopamine release probability by inhibiting ACh release from cholinergic interneurons. We also showed that M2/M4 mAChR activity had different effects on dopamine release in different subregions of the striatum (caudate-putamen versus nucleus accumbens). Threlfell S, et al. J. Neurosci. 30, 3398-3408, 2010. 5. Aberrant processing of the amyloid precursor protein (APP) is thought to play an important role in the pathophysiology of Alzheimer's disease. We found that M1 mAChR KO mice showed enhanced accumulation of amyloid plaque pathology. This observation indicates that the M1 mAChR is an important regulator of amyloidogenesis in the brain. Thus, drugs targeting the M1 mAChR may become useful in the treatment of Alzheimer's disease. Davis AA, et al. J. Neurosci. 30, 4190-4196, 2010. 6. ACh is the major neurotransmitter of the olivocochlear efferent system, which provides feedback to cochlear hair cells and sensory neurons. Studies with mAChR KO mice demonstrated that presynaptic M2 and/or M4 autoreceptors exert an inhibitory effect on ACh release from olivocochlear terminals during high-level acoustic stimulation. This finding raises the possibility that muscarinic antagonists could be employed to enhance the resistance of the inner ear to noise-induced hearing loss. Maison SF, et al. J. Neurosci. 30, 6751-6762, 2010. 7. Studies with a novel M3 mAChR knockin mouse strain indicated that M3 receptor-dependent learning and memory depends, at least in part, on M3 receptor phosphorylation and arrestin signaling. This study raises the possibility that 'biased'M3 receptor agonists that preferentially promote arrestin-dependent signaling pathways may prove beneficial in the treatment of cognitive disorders. Poulin B, et al. Proc. Natl. Acad. Sci. U.S.A. 107, 9440-9445, 2010. 8. We studied the effect of carbachol on I-Ca in single intestinal myocytes from M2 or M3 mAChR receptor knockout (KO) and wild-type littermates. This analysis showed that M2 receptors mediate a phasic I-Ca suppression via G-i/o proteins, while M3 receptors mediate a sustained I-Ca suppression via G-q/11 proteins (in intestinal myocytes). Tanahashi Y, et al. Br. J. Pharmacol. 158, 1874-1883, 2009. 9. Using lung-slice preparations from M2 and M3 receptor KO mice, we demonstrated that M2 and M3 receptor-mediated bronchoconstriction is caveolae-dependent. Since caveolin-3 was found to be directly associated with M2 receptors, it is likely that caveolin-3 as novel regulator of M2 receptor function. Schlenz H, et al. Am. J. Physiol. Lung Cell. Mol. Physiol. 298, L626-L636, 2010. 10. Using tissues from mAChR KO mice, we studied the role of the M1-M5 mAChRs in regulating the stability (integrity) of the neuromuscular junction. This analysis showed that axon terminals are unstable in the absence of M2 receptors and that muscle fiber growth is impaired in mice lacking M5 receptors. These findings should be of relevance for the development of new pharmacological approaches towards the treatment of neuromuscular disorders. Wright MC, et al.. J. Neurosci. 29, 14942-14955, 2009. 11. Studies with azoxymethane-treated mice showed that hepatocyte proliferation was reduced and gross liver nodularity, apoptosis, and fibrosis were augmented in M3 receptor KO mice, as compared to wild-type control mice. This finding suggests that the M3 mAChR plays an important role in the liver injury response by modulating hepatocyte proliferation and survival. Khurana S, et al. J. Pharmacol. Exp. Ther. 333, 639-649, 2010. 12. Studies with tissue-specific M3 mAChR KO mice demonstrated that the activity of central M3 receptors promotes bone mass accrual by decreasing the sympathetic tone. Shi Y, et al. Cell Metab. 11, 231-238, 2010.